Natural products are extremely important sources of bioactive compounds for agricultural and pharmaceutical applications. Enzymes involved in secondary metabolism hold great potential as biocatalysts that may be used in the efficient synthesis of fine chemicals and high value pharmaceuticals. In this collaborative work between a metabolic engineering group and a structural biology group, we will harvest this potential towards the one-step synthesis of the blockbuster drug simvastatin (Zocor.). Simvastatin is currently synthesized from the natural product lovastatin via inefficient, multistep processes. Our proposed biosynthesis of simvastatin will result in a completely novel process that can be an attractive alternative over the current chemical routes. The central enzyme in this study is LovD, an acyltransferase from the lovastatin biosynthetic pathway. We have performed extensive, preliminary biochemical characterization of this enzyme to show that LovD is a simvastatin synthase, and can be potentially engineered into a powerful biocatalyst for simvastatin biosynthesis. This proposal will examine the following specific aims: [unreadable] [unreadable] AIM 1: Directed Evolution of LovD. We will use directed evolution methods to improve the catalytic efficiencies of LovD towards simvastatin synthesis. We have developed a high throughput screening assay based on the formation of simvastatin. [unreadable] [unreadable] AIM 2: Structure-Based Engineering of LovD. The X-ray crystal structure of LovD will be pursued in the Yeates Lab. Rational mutagenesis of key residues identified from structural analysis will be performed to probe LovD function and improve LovD catalytic properties towards simvastatin synthesis. [unreadable] [unreadable] AIM 3: Metabolic Engineering of E. coli as a whole cell biocatalyst. We will engineer the multidrug transporter system of E. coli to improve its efficiency in exporting simvastatin to the extracellular space. This will improve the conversion of the whole cell reactor at high product concentrations. [unreadable] [unreadable] AIM 4: Direct Biosynthesis of Simvastatin from A. terreus. We will metabolically engineer A. terreus to be blocked in lovastatin biosynthesis, but robust in simvastatin biosynthesis. Project Narrative [unreadable] [unreadable] We have proposed biochemical and structural studies to investigate a simvastatin synthase recently identified from our laboratories. We will use protein and metabolic engineering methods to develop a whole cell biocatalyst that can biosynthesize simvastatin. This work will represent an important milestone in biocatalysis, application of enzymes towards the synthesis of a compound as commercially important as simvastatin has not been reported. The successful outcome of engineering a natural product biosynthetic enzyme into a useful biocatalyst may lead to additional efforts to examine this class of fascinating enzymes from a biocatalysis prospective. At the same time, the outcome of the proposed work will provide important scientific insight into protein engineering, enzyme structure and function, E. coli membrane transport, and Aspergillus metabolism. [unreadable] [unreadable] [unreadable]